Project Summary Selective loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNc) is a hallmark characteristic of Parkinson's disease (PD). PD is the most common neurodegenerative movement disorder and preferentially affects men, who have two-fold increased risk of PD incidence compared to women. Substantial evidence in humans and animal models of PD suggest estrogen is responsible for this decreased risk. How estrogen protects SNc DA neurons is not clear. Though the mechanisms underlying SNc DA neuron vulnerability are not fully understood, mitochondrial dysfunction is a clear contributor to PD pathology. SNc DA neurons are critical regulators of voluntary movement. Given the importance of voluntary movement to evolutionary survival, SNc DA neurons evolved redundant mechanisms to ensure ready calcium, neurotransmitter, and ATP sufficient to sustain prolonged release. Cav1 ca2+ channels help to establish SNc readiness by supporting pacemaking, stimulating DA synthesis, and triggering mitochondrial ATP production. Additionally, evidence suggests that dopamine metabolism via monoamine oxidase (MAO) stimulates mitochondrial ATP production. While these effects may provide short-term advantage, continual perpetual stimulation of mitochondrial respiration in SNc DA neurons generates reactive oxygen and nitrogen species, and increased mitochondrial damage and turnover. My preliminary data demonstrate that baseline mitochondrial oxidation in SNc DA axons of female mice is reduced compared to male mice. This effect was further increased by 17?-estradiol and eliminated by estrogen receptor inhibitor, 4-hydroxytamoxifen. There are two potential mediators of this effect. First, estrogen is an MAO inhibitor. Second, estrogen acutely and potently inhibits Cav1 ca2+ channels. Given this preliminary data, we hypothesize that estrogen protects SNc DA neurons by lowering axonal mitochondrial oxidant stress through two convergent mechanisms: 1) by diminishing MAO activity and 2) inhibiting Cav1 Ca2+ channels. We will test this hypothesis using two photon laser scanning microscopy in ex vivo brain slices of animals with targeted expression of fluorescent mitochondrial oxidation or calcium sensors coupled with rigorous pharmacology. Additionally, we will assess the contribution of 17?-estradiol to the bioenergetic demands of sustained dopamine release. In addition to addressing these timely and important questions, this project will provide advanced training in optical, electrophysiological, pharmacological, and genetic techniques, which, when coupled with training in scientific rigor from Drs. Surmeier and Woolley, will propel me to an independent and productive academic career.